Hybrid immunity to SARS-CoV-2 arises from serological recall of IgG antibodies distinctly imprinted by infection or vaccination

Summary We describe the molecular-level composition of polyclonal immunoglobulin G (IgG) anti-spike antibodies from ancestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, vaccination, or their combination (“hybrid immunity”) at monoclonal resolution. Infection primarily triggers S2/N-terminal domain (NTD)-reactive antibodies, whereas vaccination mainly induces anti-receptor-binding domain (RBD) antibodies. This imprint persists after secondary exposures wherein >60% of ensuing hybrid immunity derives from the original IgG pool. Monoclonal constituents of the original IgG pool can increase breadth, affinity, and prevalence upon secondary exposures, as exemplified by the plasma antibody SC27. Following a breakthrough infection, vaccine-induced SC27 gained neutralization breadth and potency against SARS-CoV-2 variants and zoonotic viruses (half-maximal inhibitory concentration [IC50] ∼0.1–1.75 nM) and increased its binding affinity to the protective RBD class 1/4 epitope (dissociation constant [KD] < 5 pM). According to polyclonal escape analysis, SC27-like binding patterns are common in SARS-CoV-2 hybrid immunity. Our findings provide a detailed molecular definition of immunological imprinting and show that vaccination can produce class 1/4 (SC27-like) IgG antibodies circulating in the blood.


Figure S1 .
Figure S1.Experimental workflow and bulk serology of a select cohort; related to Figure 1.(A) Sample and workflow schematic for vaccination-infection and infection-vaccination groups examined in this study.(B) Bulk serological spike binding (left) and viral neutralization (right) titers against ancestral (top) and Omicron BA.1 (bottom) SARS-CoV-2 viruses across six donors at each Ig-seq time point examined.Run in duplicate, error bars represent SEM about the mean.

Figure S2 .
Figure S2.Ig-Seq heatmap of IgG anti-S hybrid immunity; related to Figure 1.For each of the six donors examined by Ig-Seq, each column represents a unique plasma IgG lineage across the two time points examined, ordered by relative abundance (% anti-spike plasma IgG) at the initial time point.Colored antibody markers above each lineage indicate fulllength VH:VL pairs were identified, and recombinant antibody was expressed, with domain specificity determined by indirect ELISA against individual spike domains.Only plasma IgG lineages present at >0.5% relative abundance at the initial time point are shown.
Figure S3.Neutralization and sequence comparison of mAb clone SC27-LV (progenitor of SC27 identified in plasma post-vaccination) to SC27 (post-BT infection clone); related to Figure 3. (A) Live-virus neutralization assays using SC27 and SC27-LV screened against a panel of SARS-CoV-2 VOCs as well as zoonotic sarbecoviruses.(B) Global sequence alignment of SC27 and SC27-LV variable heavy (VH) regions.(C) Global sequence alignment of SC27 and SC27-LV variable light (VL) regions.In (B) and (C), amino acid differences are colored blue (similar) or red (dissimilar)."LV" notates "late vaccination" based on the level of somatic hypermutation within the SC27 plasma IgG lineage.

Figure
Figure S4.Cryo-EM processing summary for the spike trimer bound to SC27 Fab; related to Figure 4.

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Figure S5.Cryo-EM processing summary for the RBD-mediated dimer-of-trimers; related to Figure 4.

Figure S7 .
Figure S7.Comparative analysis of the SC27-RBD complex aligned with other published structures of mAbs targeting a similar epitope; related to Figure 4.

Figure S8 .
Figure S8.Deep mutational scanning of SARS-CoV-2 XBB.1.5RBD and SC27 mAb escape; related to Figure 4. (A) Heatmap of mutation-escape scores at key sites.Residues marked with X are the wild-type amino acids in XBB.1.5.Amino acids absent from the library are shown in grey.Complete heatmap and code for (A) can be found at https://dmsvep.github.io/SARS-CoV-2_XBB.1.5_RBD_DMS_SC27/htmls/summary_overlaid.html.(B) Surface representation of spike RBD colored by the sum of escape scores at that site.

Table S1 . Convalescent and post-vaccination donor blood sample information; related to Figure 1. In
determined at the time of the first blood draw."Inf/Vax date" refers to the date of infection, or, for the vaccines administered in two doses (Pfizer BNT162b2 and Moderna mRNA 1273), the date of the first vaccine dose, with a second dose being administered ~21 days later."Days since previous S exposure" refers to the days between infection and vaccination, in either order.The "Gender/Age" column indicates age at the time of study recruitment.

Table S4 . PISA analysis of interface between SC27 Fab and Omicron BA.1 spike protein; related to Figure 4
. "ASA" = "accessible surface area"."BSA" = "buried surface area"."% BSA" = percentage of buried surface area for each residue within the interface.Only residues with interface BSA >0 are listed.All surface area values are Å 2 .